Antioxidant composition

ABSTRACT

The present invention relates to an antioxidant composition comprising a combination of galatomannan and N-acetyl cysteine for its use in the treatment of a skin disease or condition resulting from reactive oxygen species production in the skin or involving reactive oxygen species production in the skin, to a hydrogel containing said combination as well as to dressing wounds comprising said hydrogel and its use in the healing of ulcers, wounds, burns and scalds.

FIELD OF THE INVENTION

The present invention relates to antioxidant compositions and their usein the treatment of diseases, disorders and conditions affecting theskin, particularly skin conditions that involve the production ofreactive oxygen species in human skin, such as photoaging and otherage-related skin damage. It also refers to wound dressings andparticularly to compositions for delivery active agents to wounds.

BACKGROUND

The aging of the population and the increase of chronic metabolicdiseases such as hypertension or diabetes has favored the presence ofchronic ulcers in recent years.

Occasionally, due to the health condition of the patient (diabetes) orto the presence of a large amount of damage in the tissue (seriousburns), phenomena can arise which alter the series of processes whichmust take place for healing to occur, developing a chronic ulcer.

The formation of a chronic ulcer is associated with an excessiveinflammatory process which alters the synthesis of the signalingmolecules involved in regulating the process taking place in healing.Recent studies directly relate the physiopathology of chronic ulcerswith the oxidative stress present in the bed of the wound, as aconsequence of the proinflammatory environment of the lesioned area.

When oxidative stress occurs, the organism has detoxification mechanismscapable of control the excess of the reactive oxygen species generated,in contrast, when there is a lack of adjustment between the detoxificantcapacity of the organism and the free radicals present in the bed of thewound, the healing process slows down, giving rise to a chronic ulcer.

A lack of adjustment in the detoxification mechanisms of reactive oxygenmetabolites is one of the main causes of chronicity in ulcers.

The healing of chronic wounds may be induced by the use of antioxidantwound dressings that react specifically with excess reactive oxygenspecies and hence reduce the level of oxidative stress. In the art,different examples of materials for healing purposes are described.

For example, in U.S. Pat. No. 6,406,712, a wound dressing material hasbeen described, which is formed by mixing dry hydrocolloid polymerpowder with water contained in a sealed package having a temporary ormanually-removable barrier so that the dry polymer and water can bestored separately from each other while in the package.

Another description, at the patent application WO 01/49258A2, comprisestissue contact materials, such as biocompatible polymer comprising anon-gellable polysaccharide, such as guar gum, that entrap oxygen withinclosed cell foam-like material capable of providing or maintainingoptimal oxygen tension at a compromised tissue site while absorbingexcess fluid and optimizing the microenvironment to facilitate tissuerepair and regeneration if needed.

The patent application EP0781550A1, describes a bioadhesivepharmaceutical composition for the controlled liberation of activeingredients, antiulcer among others, constituted by a co-polymer ofvinyl acetate and polyvinylpyrrolidone and an additional component, suchas the locust bean gum among others.

The antioxidant activity of galactomannans upon reducing the lipidperoxidation of systems subjected to UVA radiation has also recentlybeen described. Their capacity to increase the elasticity of differentmixtures of hydrogels and their capacity to absorb water, being able toprovide the bed of the wound with the necessary degree of moistureneeded by the healing process, are also known.

The international application WO2005/084650A1 claims a storage stableand dry active ingredient delivery system for pharmaceutically activeingredients for dermal use with wound healing purposes. The deliverysystem comprises a xerogel in that the gel-forming material is apolysaccharide, for example galactomannan derivatives. When the xerogelcomes into contact with fluids it is rehydratated and forms a hydrogel,whereby the applied active ingredients are dissolved and released at acontrolled rate from the hydrogel leading to a locally highconcentration.

Solid, bioabsorbable materials for use as wound dressings are describedat the patent application EP0792653, where such a solid is formed by amixture of xanthan, and at least one galactomannan, such as guar gum orlocust bean gum. The material also comprises therapeutic agents amongwhich are particularly preferred those that actively promote woundhealing such as glicosaminoglycans.

In a similar procedure as described above a wound dressing with healingpurposes is claimed by the international application WO99/25395, wherethe matrix comprises a biocompatible cross-linked polymer and anon-gellable polysaccharide, a galactomannan, which includes as well oneor more active ingredients, for example, wound healing agents likegrowth factors, mucopolysacharides and proteins.

Other types of wound healing dressings are described by theinternational applications WO2004/112850 and WO2005/049101 wheregenerally the material is formed by a bioabsorbable substrate, whichcould be galactomannan, dyed with an antioxidant dyestuff, which canreact with oxygen reactive species, reducing, in that way, the level ofoxidative stress at the wound.

N-acetyl cysteine is also known as an antioxidant molecule which acts byincreasing the synthesis of intracellular glutation (GSH). The reducingeffect of GSH contributes to directly eliminating the reactive oxygenspecies and also to recycling already used antioxidants. Its use inchronic ulcers would reduce the oxidative stress thereof, thus favoringtheir healing (Manikandan, P. et al, Molecular and CellularBiochemistry, 2006, 290, 87-96; Rani Thaakur, S. et al,Pharmacologyonline, 2009, 1, 369-376).

The antioxidant activity of curcumin is also known. Curcumin is thepurified state of the raw extract of Turmeric root, a plant mostlycultivated in Southeast Asia and widely used in traditional medicine forthe treatment of skin-related diseases. Gopinath, D. (Biomaterials,2004, 25, 1911-1917) demonstrates the improved capacity of wound healingby curcumin antioxidant when it is incorporated to a collagen matrix,which also acts as a supportive matrix for the regenerative tissue.

Although the antioxidant properties of galactomannans and N-acetylcysteine are well-documented in the prior art, there is no indicationabout the particular advantages conferred by the combination of bothcomponents, and particularly, to the antioxidant synergistic effectprovided on cells cultures suffering extensive oxidative stress.

BRIEF DESCRIPTION OF THE INVENTION

The authors of the present invention have found that the combination ofgalactomannan and N-acetyl cysteine provides a synergistic effect in theantioxidant capacity of both components, which results in an unexpectedadvantage for its use in the treatment of diseases or disordersresulting from reactive oxygen species production in the skin of asubject or diseases or conditions which involves the reactive oxygenspecies production in the skin of a subject.

The experimental tests have shown that human skin cells (fibroblasts)subjected to an oxidative stress undergo an increase in the cellsurvival capacity/preservation when a combination of galactomannan, suchas locust bean gum, and N-acetyl cysteine is administered to the culturecells.

Additional experiments have also revealed that said combination providesa significant reduction in the intracellular levels of reactive oxygenmetabolites in fibroblast cell cultures subjected to an oxidative stressin the presence of oxygen peroxide, when compared to locust bean gum orN-acetyl cysteine alone.

Furthermore, an even higher synergistic effect has been observed whencurcumin (or turmeric) is added to the combination of galactomannan andN-acetyl cysteine.

Therefore, in a first aspect the present invention refers to anantioxidant composition comprising galactomannan and N-acetyl cysteinefor its use in the therapeutic or prophylactic treatment of a skindisease or condition resulting from reactive oxygen species productionin the skin of a subject or of a skin disease or condition whichinvolves the reactive oxygen species production in the skin of asubject.

Particularly preferred is the use of locust bean gum as galactomannan inthe composition used in the invention.

In a particular embodiment, the antioxidant composition as defined abovefurther comprises curcumin (turmeric) as an additional antioxidantingredient.

The invention also relates to a method for the therapeutic orprophylactic treatment of a skin disease or condition resulting fromreactive oxygen species production in the skin of a subject or of a skindisease or condition which involves the reactive oxygen speciesproduction in the skin of a subject, which comprises the administrationof a therapeutically effective amount of a composition comprisinggalactomannan and N-acetyl cysteine.

Another aspect of the present invention relates to an antioxidantcomposition which comprises galactomannan, N-acetyl cysteine andcurcumin.

Another aspect of the present invention relates to the antioxidantcomposition as defined above for its use as a medicament.

Another aspect of the invention refers to a hydrogel which comprisesgalactomannan and N-acetyl cysteine, wherein the galactomannan is in theform of a cross-linked matrix, and N-acetyl cysteine is incorporated insaid cross-linked matrix of galactomannan. In a particular embodiment,the galactomannan is cross-linked by means of a cross-linking agent,preferably the cross-linking agent is glutaraldehyde.

In another aspect, the invention relates to the hydrogel as definedabove, wherein the galactomannan matrix further comprises curcuminincorporated therein.

Additionally, the present invention also refers to the hydrogel asdefined above which further includes cells. Particularly preferred arecells selected from the group consisting of fibroblasts, keratinocytes,endothelial cells, differentiated or undifferentiated mesenchymal stemcells, corneal cells, epithelial cells, cells from leucocitary system,cells from hematopoietic system, differentiated or undifferentiated stemcells, chondrogenic cells, osteoblasts, miocytes, adipocytes and neuronsor other cells from the peripheric or central nervous system.

An additional aspect of the present invention refers to a process forthe preparation of a hydrogel as defined above which comprises:

-   -   a) dissolving the galactomannan in an aqueous solution;    -   b) subjecting the galactomannan to a chemical cross-linking by        adding a cross-linking agent to the aqueous solution of        galactomannan to obtain a hydrogel comprising a cross-linked        glucomannan matrix;    -   c) incorporating N-acetyl cysteine, and optionally the curcumin,        into the cross-linked glucomannan matrix.

Another aspect of the present invention refers to a wound dressingcomprising the hydrogel as defined above.

An additional aspect of the present invention relates to a hydrogel asdefined above for its use as a medicament.

Another aspect of the invention refers to a hydrogel as defined abovefor its use in the treatment and/or healing of acute surgical andtraumatic wounds, burns, scalds, fistulas, venous ulcers, arterialulcers, pressure sores (otherwise known as decubitus ulcers), diabeticulcers, ulcers of mixed aetiology, and other chronic or necrotic woundsand inflammatory lesions and disorders.

An additional aspect of the invention relates to cosmetic compositionwhich comprises galactomannan and N-acetyl cysteine.

In another aspect, the invention relates to the cosmetic composition asdefined above, which further comprises curcumin.

Another aspect of the present invention refers to the use of a cosmeticcomposition as defined above for the treatment of an age-related skindamage.

Another aspect of the present invention refers to the use of a cosmeticcomposition as defined above as an UV-radiation protector.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows: (a) the results of cell proliferation in fibroblasts bymeans of the MTT colorimetric assay, using different concentrations ofNAC, and (b) the IC₅₀ values with respect to the control.

FIG. 2 shows: (a) the results of cell proliferation in fibroblasts bymeans of the MTT colorimetric assay, using different concentrations ofturmeric, and (b) the IC₅₀ values with respect to the control.

FIG. 3 shows the results corresponding to the MTT colorimetric assaywhen a fibroblasts culture is subjected to an oxidative environment andwhen it is put in contact with 1% LBG, 1 mM NAC, 1 μM Turmeric andcombinations thereof.

FIG. 4 shows the intracellular ROM levels of fibroblasts subjected to anoxidative environment using 1 mM of H₂O₂, by means of the fluorescenceunits obtained in the labeling with the probe 2′,7′-dichlorofluoresceindiacetate and when fibroblasts are put in contact with 1% LBG, 5 mM NAC,5 μM Turmeric and combinations thereof.

FIG. 5 shows photographs taken from scanning electron microscope (SEM)of a hydrogel of locust bean gum cross-linked with glutaraldehyde at:(a) 0% wt; (b) 0.5% wt; (c) 1% wt and (d) 2.5% wt.

FIG. 6 shows a macroscopic view of a 10-days evolution biopsy of skinlesions surgically generated in the dorsal area in a healing animalmodel in pigs.

FIG. 7 shows a 3-days evolution photograph of skin lesions surgicallygenerated in the dorsal area in a healing animal model in pigs.

DETAILED DESCRIPTION OF THE INVENTION

The antioxidant composition used in the invention comprises twoantioxidant agents, namely a galactomannan and N-acetyl cysteine. Thesecomponents are physically mixed in the composition without being bondedby any chemical bond or interaction.

As shown in the experimental tests, the combination of a galactomannan,such as carob locust bean gum, and N-acetyl cysteine provides anantioxidant synergistic effect over fibroblast cell cultures subjectedto an oxidative stress, improving the cell survival capacity whilereducing the intracellular levels of reactive oxygen metabolites.

Galactomannans are polysaccharides containing a mannose backbone withgalactose side groups, more specifically a (1-4)-linkedbetha-D-mannopyranose backbone with branch points from their 6 positionslinked to alpha-D-galactose, i.e. 1-6 linked alpha-D-galactopyranose.Galactomannan gums include locust bean gum (LBG), guar gum, cassia gum,tara gum, mesquite gum, and fenugreek gum.

In a particular embodiment, the galactomannan is selected from the groupconsisting of guar gum, locust bean gum, cassia gum, tara gum, mesquitegum, fenugreek gum and white clover bean gum. More preferably, thegalactomannan is locust bean gum. Locust bean gum is a galactomannanpolysaccharide consisting of mannopyranose backbone with branchpointsfrom their 6-positions linked to α-D-galactose residues. Locust bean gumhas about 4 mannose residues for every galactose residue (amannose/galactose ratio of about 4).

Galactomannans may be derived from recombinant or synthetic sources. Forexample, galactomannose may be synthesized in vivo from GDP-mannose andUDP-galactose by the enzymes mannan synthase and galactosyltransferase.DNA coding for these proteins has been isolated and characterized, (USPublication 2004/0143871) and recombinant plants transformed with theseenzymes have been shown to express elevated levels of galactomannan. Inaddition, the degree of galactosylation of the mannopyranose backbonemay be influenced by the presence (or absence) of alpha-galactosidase invivo, (see Edwards et al. Plant Physiology (2004) 134: 1153-1162).Alpha-galactosidase removes galactose residues from the mannopyranosebackbone. For example, seeds that naturally express galactomannans witha lower degree of galactosylation may express (or express more) alphagalactosidase, which removes galactose moieties from the mannopyranosebackbone in those species of plant. The alpha-galactosidase enzyme maybe used to reduce the presence of galactose on the mannopyranosebackbone of naturally occurring galactomannose gums in a laboratorymanipulation of the characteristics of the naturally occurringgalactomannose gum. Embodiments of the present invention includegalactomannans, which have been treated with alpha-galactosidase toreduce the presence of galactose on the mannopyranose backbone.Embodiments of the present invention include galactomannan gums whichhave been treated with alpha-galactosidase or other enzymes or chemicaltreatments, to “tune” the gums to provide the gum with desiredcharacteristics as cell culture surfaces.

The weight proportion of galactomannan in the composition of theinvention ranges from 1 to 5% with respect to the total weight of thecomposition.

N-acetyl cysteine is an antioxidant molecule which intervenes in thesynthesis of intracellular glutathione, a compound which contribute todirectly eliminate the free oxygen radicals in the cell, as well as torecycle antioxidants already used.

The N-acetyl cysteine is preferably present in the composition of theinvention in a concentration that ranges from 1 to 10 mM, morepreferably from 1 to 5 mM.

In a preferred embodiment, the antioxidant composition used in theinvention is suitable for topical application on the skin. The topicalantioxidant compositions may take any of a wide variety of forms, andinclude, for example dressings, lotions, solutions, sprays, creams,gels, ointments, or the like.

Lotions are preparations to be applied to the skin surface withoutfriction, and are typically liquid or semi-liquid preparations in whichsolid particles, including the active ingredients, are present in awater or alcohol base. Lotions are usually suspensions of solids, andpreferably comprise a liquid oily emulsion of the oil-in-water type.Lotions are preferred formulations for treating large body areas becauseof the ease of applying a more fluid composition. It is generallypreferred that that the insoluble matter in a lotion (hydrogel) befinely divided. Lotions contain from about 0.001% to about 30% of theactive ingredients, from 1% to 25% of an emollient and the appropriateamount of water. Examples of emollients are hydrocarbon waxes and oilssuch as mineral oils, petrolatum, paraffin, ceresin, microcrystallinewax, polyethylene and perhydrosqualene; silicone oils such asdimethylpolysiloxanes, methylphenylpolysiloxanes and water-soluble andalcohol-soluble glycol-silicone copolymers; triglycerides, such asanimal and vegetable fats and oils; alkyl esters of fatty acids having10 to 20 carbon atoms, alkenyl esters of fatty acids having 10 to 20carbon atoms; fatty acids having 10 to 20 carbon atoms, such aspelargonic, lauric, myristic, palmitic, stearic, isostearic,hydroxystearic, oleic, linoleic, ricinoleic, arachidonic, behenic anderucic acids; fatty alcohols having 10 to 20 carbon atoms, such aslauryl, myristoyl, palmitoyl, stearyl, isostearyl, hydroxystearyl,oleyl, ricinoleyl, behenyl, erucyl and 2-octyl dodecanol alcohols areappropriate examples of fatty alcohols; fatty alcohol ethers, such asethoxylated fatty alcohols having 10 to 20 carbon atoms includinglauryl, cetyl, stearyl, isostearyl, oleyl and cholesterol alcoholshaving attached thereto from 1 to 50 ethylene oxide groups or 1 to 50propylene oxide groups; lanolin and derivatives; waxes such as beeswax,spermaceti, myristoyl myristate and stearyl stearate; beeswaxderivatives, such as polyoxyethylene sorbitol beeswax; vegetable waxes,including, but not limited to, carnauba and candelilla waxes;phospholipids such as lecithin and derivatives; sterols, such ascholesterol and acyl esters of cholesterol; and amides, such as fattyacid amides, ethoxylated acyl amides and solid fatty acid alkanolamides.

The lotions of the invention would further contain from 1% to 30% of anemulsifier. The emulsifiers can be anionic, cationic or non-ionic.Examples of non-ionic emulsifiers include, but are not limited to, fattyalcohols having 10 to 20 carbon atoms, fatty alcohols having 10 to 20carbon atoms condensed with 2 to 20 moles of ethylene oxide or propyleneoxide, alkyl phenols with 6 to 12 carbons in the alkyl chain condensedwith 2 to 20 moles of ethylene oxide, mono- and di-acyl esters ofethylene glycol, wherein the fatty acid contains from 10 to 20 carbons,monoglycerides wherein the fatty acid contains from 10 to 20 carbons,diethylene glycol, polyethylene glycols of molecular weight 200 to 6000,polypropylene glycol of molecular weight 200 to 3000, glycerol,sorbitol, sorbitan, polyoxyethylene sorbitol, polyoxyethylene sorbitanand hydrophilic wax esters. Suitable anionic emulsifiers include, butare not limited to, fatty acids saponified (soaps) with potassium,sodium, or triethanolamine, wherein the fatty acid contains from 10 to20 carbons. Other suitable anionic emulsifiers include, but are notlimited to, alkali metals, ammonium or substituted ammonium with alkylsulfates, alkyl arylsulfonates and alkyl ethoxy ether sulfonates having10 to 30 carbons in the alkyl chain and from 1 to 50 ethylene oxideunits. Suitable cationic emulsifiers include quaternary ammonium andmorpholinium and pyridinium compounds.

The balance of the composition is water. The lotions are formulated bysimply admixing all of the components together. Preferably, the activeingredients are dissolved in the emollient and the resulting mixture isadded into the water.

The compositions of the present invention may also be formulated in theform of a solution. Solutions are homogenous mixtures prepared bydissolving the active ingredients in a liquid such that the molecules ofthe dissolved ingredients are dispersed among those of the solvent.Solutions contain from 0.001% to 30% of the antioxidant activeingredients and the adequate amount of an organic solvent. Organicsubstances useful as the solvent are propylene glycol, polyethyleneglycol, polypropylene glycol, glycerine, sorbitol esters,1,2,6-hexanetriol, ethanol, isopropanol, diethyl tartrate, butanediol,and mixtures thereof. Such solvent systems can also contain water.

These compositions are applied on the skin in the form of a solution, orsolutions are formulated in the form of aerosol and applied on the skinas a spray.

Compositions in the form of aerosol additionally contain from 25% to 80%of a suitable propellant. Examples of propellants include, but are notlimited to chlorinated, fluorinated and fluorochlorinated low molecularweight hydrocarbons. Nitrous oxide and carbon dioxide are also used aspropellant gases. Enough quantity to expel the content of the cartridgeis used.

The composition of the present invention may be also formulated in theform of a cream. For instance, creams, as is well known in the arts ofpharmaceutical and cosmetic formulations, are viscous liquids orsemisolids emulsions, either oil-in-water or water-in-oil. Cream basesare water-washable, and contain an oil phase, an emulsifier, and anaqueous phase. The oil phase is generally comprised of petrolatum and afatty alcohol such as cetyl or stearyl alcohol. The aqueous phaseusually, although not necessarily, exceeds the oil phase in volume, andgenerally contains a humectant. The emulsifier in a cream formulation isgenerally a nonionic, anionic, cationic or amphoteric surfactant and canbe selected from emulsifiers mentioned above for lotions or mixturesthereof.

Gels are semisolid, suspension-type systems. Single-phase gels containorganic macromolecules distributed substantially uniformly throughoutthe carrier liquid, which is typically aqueous, but also, preferably,contain an alcohol, and, optionally, an oil. Preferred organicmacromolecules, i.e. gelling agents, may be chemically crosslinkedpolymers such as crosslinked acrylic acid polymers, for instance the“carbomer” family of polymers, e.g., carboxypolyalkylenes, that may beobtained commercially under the Carbopol® trademark. Also preferred maybe hydrophilic polymers such as polyethylene oxides,polyoxyethylene-polyoxypropylene copolymers and polyvinylalcohol;cellulosic polymers such as hydroxypropyl cellulose, hydroxyethylcellulose, hydroxypropyl methyl cellulose and methyl cellulose; gumssuch as tragacanth and xanthan gum; sodium alginate; and gelatin.

Ointments, as also well known in the art, are semisolid preparationsthat are typically based on petrolatum or other petroleum derivatives.The specific ointment base to be used, as will be appreciated by thoseskilled in the art, is one that will provide for a number of desirablecharacteristics, e.g., emolliency or the like. Ointment bases may begrouped in four classes: oleaginous bases, emulsifiable bases, emulsionbases and water-soluble bases. Oleaginous ointment bases include, forexample, vegetable oils, fats obtained from animals, and semisolidhydrocarbons obtained from petroleum. Emulsifiable ointment bases, alsoknown as absorbent ointment bases, contain little or no water andinclude, for example, hydroxystearin sulfate, anhydrous lanolin, andhydrophilic petrolatum. Emulsion ointment bases are either water-in-oilemulsions or oil-in-water emulsions, and include, for example, cetylalcohol, glyceryl monostearate, lanolin, and stearic acid. Preferredwater soluble ointment bases are prepared from polyethylene glycols ofvarying molecular weight.

A pharmaceutical acceptable vehicle may also be incorporated in thecompositions and may be any vehicle conventionally used in the art.Examples include water, lower alcohols, higher alcohols, polyhydricalcohols, monosaccharides, disaccharides, polysaccharides, hydrocarbonoils, waxes, fatty acids, silicone oils, nonionic surfactants, ionicsurfactants, silicone surfactants, and water-based mixtures andemulsion-based mixtures of said vehicles.

Topical compositions described above may be applied regularly towhatever skin area that requires treatment with the frequency and in theamount necessary to achieve the desired results. The frequency oftreatment depends on the nature of the skin disease or condition, i.e. askin disease or condition that results from ROS production in skin, aswell as the degree of damage or deterioration of the skin.

Due to the antioxidant properties of the combination of galactomannanand N-acetyl cysteine, it can be used to treat or prevent a skin diseaseor condition that results from reactive oxygen species production in theskin of a subject, or of a skin disease or condition which involves thereactive oxygen species production in the skin of a subject,particularly in skin fibroblasts and keratinocytes.

This treatment includes contacting the skin of a subject by directlyapplying to the skin a topical formulation as herein described, in amanner that affects the subject, and/or skin tissue in the subjectand/or one or a plurality of cells, to obtain a desired pharmacologiceffect and/or physiologic effect. The effect may be prophylactic interms of completely or partially preventing a disease or disorder suchas a condition that results from reactive oxygen species production inskin or that involves reactive oxygen species production in skin, or asign or symptom thereof, and/or the effect may be therapeutic in termsof relieving symptoms or signs or providing a partial or complete curefor such a disorder or disease and/or substantially impairing an adverseeffect attributable to the disorder or disease.

Related embodiments, contemplate, by way of example:

-   -   (i) preventing the disease or disorder (e.g., skin condition        that results from oxygen reactive species production or that        involves oxygen reactive species production) from occurring in a        subject that may be predisposed to the disease or disorder, but        has not yet been diagnosed as having it;    -   (ii) inhibiting the disease or disorder, i.e., arresting its        progression; or    -   (iii) relieving or ameliorating the disease or disorder, i.e.        causing regression.

In a particular embodiment, the treatment of a skin disease or conditionthat results from the production of reactive oxygen species includes therepair and regeneration of damaged or injured tissue or cells at a siteof skin damage. This damage can be the result of the exposure of thesubject to a source of oxidative stress that may promote radical oxygenspecies production in skin, such as sunlight radiation (photodamage),chemical agents (including other topical agents such as medical,pharmaceutical or cosmetic compounds), radiotherapy or chemotherapy. Italso includes prophylactic treatments to prevent such damage, forinstance, prior to exposure of the subject to a source of oxidativestress that may promote radical oxygen species production in skin, suchas UV radiation, chemical agents (including other topical agents such asmedical, pharmaceutical or cosmetic compounds) or prior to radiotherapyor chemotherapy.

More particularly, the skin disease or condition results from theexposure to sunlight, more specifically to the UV radiation of type UVA,UVB and UVC. Conditions directly or indirectly a consequence of (or areexacerbated by, or include as a risk factor) exposure to such radiationinclude both direct and immediate effects, as well as longer termeffects, and complications and sequellae that arise from the directdamage, over a longer term.

It is thought that UV radiation impacts skin through both direct andindirect mechanism. The direct damage is that which is incurred uponimmediate exposure to radiation, whereas the indirect effects includethose which follow the generation of damaged biological molecules andthe generation of highly reactive oxygen species which then set otherbiological and pathological processes in motion. The reactive oxygenspecies may have deleterious effects in the immediate locale where theyare generated, as in the skin, or at distant sites, where such reactivespecies may have broader systemic effects, as may manifest in what istermed “oxidative stress”. An intervention that effectively reduces thelevel of reactive species, thereby having an anti-oxidant effect, thusmay have slow, ameliorate, or stop the progression of a broad range ofdiseases.

Health problems associated with exposure to UV radiation involveconditions or diseases of the skin, but more widespread and systemicconditions may also arise, or be a part of complications that follow onas a consequence of such conditions or diseases of the skin.Accordingly, such conditions, collectively, may include sunburn,photosensitivity, immunosuppression, premature aging, psoriasis, severaltypes of skin cancer and various immunological diseases, as well aslocalized or widespread inflammation, various bacterial or fungalinfections, skin rashes, and systemic oxidative stresses caused by UVradiation exposure and diet. Actinic keratosis, for example, is aprecancerous lesion developed after many years of sun exposure.Polymorphic light eruption, for example, is a rash induced by sunlightexposure, which is understood as involving skin-localized allergy. Typesof skin cancer linked to sunlight exposure include, in order ofincreasing seriousness, basal cell cancer, squamous cell cancer, andmalignant melanoma.

In another particular embodiment, the skin disease or condition whichinvolves the reactive oxygen species production in the skin of a subjectis selected from acute surgical and traumatic wounds, burns, scalds,fistulas, venous ulcers, arterial ulcers, pressure sores (otherwiseknown as decubitus ulcers), diabetic ulcers, ulcers of mixed aetiology,and other chronic or necrotic wounds and inflammatory lesions anddisorders.

In a particular embodiment of the present invention, the antioxidantcomposition of the invention further comprises curcumin as an additionalactive ingredient. It has been found that the combination ofgalactomannan with N-acetyl cysteine and curcumin provides an evenhigher synergistic antioxidant effect as shown the examples provided inthe present application.

Cucumin, also known as turmeric, is a naturally occurringo-methoxyphenol derivative of formula:

It is a yellow pigment obtained from the rizhomes of Curcuma Longa andit has been used for centuries in indigenous medicine for the treatmentof a variety of inflammatory conditions. Curcumin also acts as a freeradical scavenger and antioxidant, inhibiting lipid peroxidation andoxidative DNA damage.

The curcumin is preferably present in the composition of the inventionin a concentration ranging from 1 to about 7.5 μM, more preferably from1 to 5 μM.

Therefore, another aspect of the present invention refers to anantioxidant composition which comprises glactomannan, N-acetyl cysteinaand curcumin.

Said antioxidant composition is also suitable for topical application onthe skin and may take any of a wide variety of forms, including, forexample dressings, lotions, solutions, sprays, creams, gels, ointments,or the like, such as those mentioned above.

Additionally, the present invention relates to the antioxidantcomposition which comprises glactomannan, N-acetyl cysteine and curcuminfor its use as a medicament.

This antioxidant composition can also be used to treat or prevent a skindisease or condition that results from reactive oxygen speciesproduction in the skin of a subject or a skin disease or condition whichinvolves the reactive oxygen species production in the skin of asubject, such as those mentioned above.

In another particular embodiment, the present invention relates to ahydrogel which comprises galactomannan and N-acetyl cysteine, whereinthe galactomannan is in the form of a cross-linked matrix and N-acetylcysteine is incorporated in said cross-linked matrix of galactomannan.

The term “hydrogel” refers to a network of polymer chains whichcomprises cross-linked galactomannan chains that are water-insoluble butwater-swellable, i.e. the water is the dispersion medium.

The hydrogel of the invention provides a reliable and efficient meansfor delivery N-acetyl cysteine to the site of interest, such as to awound, ulcer, burn or scald, while improving the antioxidant and healingproperties of this active ingredient. Indeed, the experimental testshave shown the antioxidant synergistic effect induced by the combinationof a galactomannan, such as locust bean gum, and N-acetyl cysteine overfibroblast cell cultures, improving the cell survival capacity whilereducing the intracellular levels of reactive oxygen metabolites. Thehydrogel also provides a very good moisture regulation capacity forpromoting wound healing.

The hydrogel of the invention comprises polymerized chains ofgalactomannan, said chains of galactomannan are cross-linked in order tomake galactomannan water-insoluble but water-swellable. Thecross-linking degree determines the reological properties of thehydrogel, as well as its swellable properties, and allows obtaining aporosity that favors the controlled delivery of N-acetyl cysteine.

Particularly, the galactomannan is selected from the group consisting ofguar gum, locust bean gum, cassia gum, tara gum, mesquite gum, fenugreekgum and white clover bean gum. More preferably, the galactomannan islocust bean gum.

In a particular embodiment, galactomannan is cross-linked by means of across-linking agent. Chemical agents such as borax (sodium borohydrate),glutaraldehyde and epoxy derivatives can be used. Particularly, the mostpreferred cross-linking agent is glutaraldehyde.

The cross-linking agent content determines the pore size of the matrixand thus the delivery profile of the active ingredient incorporatedtherein.

The galactomannan may be present in the hydrogel according to theinvention in an amount of at least 50% by weight with respect to thetotal weight of the hydrogel, preferably at least 75% by weight. Morepreferably, at least 90% by weight of the hydrogel consists ofgalactomannan.

The balance of the hydrogel comprises water (up to 20% by weight), theactive ingredient (N-acetyl cysteine) and, optionally, salts or otherstructural compounds which improve the reological properties of thehydrogel.

Among the structural compounds which can be optionally present in thehydrogel, proteins such as collagen, fibronectin, laminin, elastin orcombinations thereof, as well as glycosaminoglycans, such ashyaluronates, heparin sulfate or chondroitin sulfate, are preferred.

Preferably, the hydrogel according to the present invention will absorbwater or wound fluid and hence become wet, swell or become gelatinousmass but will not spontaneously dissolve or disperse therein. Lowsolubility renders such materials especially suitable for use as wounddressings to remove reactive oxygen species from the wound fluid.

N-acetyl cysteine may be directly incorporated into the cross-linkedgalactomannan matrix. This active ingredient may be incorporated byabsorption of the agent by the matrix or by adding the agent into theinitial formulation for the matrix prior to cross-linking.

In a preferred embodiment of the invention, the incorporation ofN-acetyl cysteine into the galactomannan matrix is carried out by theformation of a xerogel.

The term “xerogel” refers to a solid substrate formed from a hydrogel bydrying with unhindered shrinkage. It retains high porosity (at least25%) and enormous surface area (150-900 m²/g) along with very small poresize (1-10 nm).

The obtained xerogel is introduced in an aqueous solution comprisingN-acetyl cysteine and then, this active ingredient is graduallyincorporated into the porous of the matrix or dispersed therein untilthe equilibrium is reached.

The N-acetyl cysteine is preferably present in the hydrogel in aconcentration ranging from 1 to 10 mM, more preferably from 1 to 5 mM.

Another aspect of the present invention refers to the hydrogel of theinvention mentioned above which further comprises curcumin as anadditional active ingredient to be incorporated in the matrix ofgalactomannan. It has been found that the combination of agalactomannan, such as locust bean gum, with N-acetyl cysteine andcurcumin provides an even higher synergistic antioxidant effect.

As in the case of N-acetyl cysteine, curcumin may also be incorporatedinto the galactomannan matrix by absorption of this compound by thematrix or by adding it in the initial formulation for the matrixtogether with N-acetyl cysteine prior to cross-linking thegalactomannan.

However, it is also preferred to incorporate curcumin and N-acetylcysteine by introducing a xerogel of galactomannan into a solutioncomprising both active ingredients, thus allowing the gradualincorporation thereof into the matrix of galactomannan.

Curcumin is preferably present in the hydrogel in a concentrationranging from about 1 to about 7.5 μM, more preferably from 1 to 5 μM.

It is to be understood that the active ingredients are incorporated intothe hydrogel, so that the agents are released directly from the hydrogeland further delivered via transdermal or transmucosal pathways. Theincorporated agents may be released over an extended period of time inorder to facilitate wound healing.

In a particular embodiment, once the active ingredient(s) areincorporated and dispersed throughout the matrix of glucomannan, aportion of the agent resides in the matrix while the other portion ofthe agent is dissolved in the free liquid phase and moves freely throughthe matrix. Because the agent is dissolved in the free liquid phase, aconcentration gradient of the active agent is created between the matrixof the hydrogel and the moisture of the wound itself. Therefore, whenthe hydrogel is placed onto a moist surface such as an open wound, thesoluble agent will move through the free liquid phase toward theagent-free wound moisture, resulting in the delivery of the agent to thewound. This movement of soluble agent further upsets the equilibriumbetween soluble and insoluble agents, and causes more agent to dissolveinto the free liquid phase, thus causing more agent to be delivered tothe wound.

Delivery of the active ingredients may also be controlled by the degreeof cross-linking in the matrix. The combination of chains cross-linkedtogether creates microcavities wherein the active ingredients areencapsulated. By controlling the amount of cross-linking agent and thelength of galactomannan chains, it is possible to regulate the size ofthe microcavities of the galactomannan matrix. Larger microcavitiesproduced by a lower degree of cross-linking, allow for freer migrationand quicker delivery of the active agents, whereas smaller microcavitiesincrease the delivery time.

The process for the preparation of the hydrogel of the inventioncomprises:

-   -   a) dissolving the galactomannan in an aqueous solution;    -   b) subjecting the galactomannan to a chemical cross-linking by        adding a cross-linking agent to the aqueous solution of        galactomannan to obtain a hydrogel comprising a cross-linked        glucomannan matrix;    -   c) incorporating N-acetyl cysteine, and optionally the curcumin,        into the cross-linked glucomannan matrix.

Preferably, the galactomannan is dissolved in distilled water at roomtemperature in an amount ranging from 1% to 5% by weight with respect tothe total weight of the solution. This solution is maintained understirring for approximately 2-3 hours. Depending on the galactomannan, itmay be required to increase the temperature in order to facilitate thedissolution thereof.

In a particular embodiment, the galactomannan is locust bean gum. Inthis case, the dissolution should be done at a temperature between 110and 120° C.

The cross-linking step is carried out with the aim of forming atridimensional matrix structure, providing it with porous or cavitieswherein the active ingredient will be incorporated. Cross-linkingmethods include UV-induced cross-linking and chemical cross-linking.Chemical agents such as borax (sodium borohydrate), glutaraldehyde,epoxy derivatives and other methods known in the art can be used. UVcross-linking methods require a photoinitiator that initiates thegelling or cross-linking process upon exposure of UV radiation.

The cross-linking degree depends on the amount of cross-linking agentadded to the solution and it ranges from about 1% to about 5% by weightwith respect to the total weight of the aqueous solution. Preferably,the cross-linking agent is glutaraldehyde.

In a particular embodiment, the solution of galactomannan and thecross-linking agent in maintained under stirring for at least 30minutes. Subsequently, the solution is poured into molds, maintainingtherein until the formation of the hydrogel. The non-reactingcross-linking agent is removed by several washes.

The incorporation of the N-acetyl cysteine, and curcumin when thisactive ingredient is present in the formulation of the hydrogel, may bedone by absorption of the agent by the matrix. Alternatively, the activeingredient(s) may be added to the aqueous solution of galactomannanprior to the cross-linking thereof.

In a preferred embodiment of the invention, the incorporation of theN-acetyl cysteine, and optionally the curcumin, comprises the followingsteps:

-   -   1) drying the hydrogel obtained in step b) to form a xerogel;    -   2) rehydrating the xerogel by introducing it in an aqueous        solution comprising N-acetyl cysteine, and optionally curcumin,        to form a hydrogel wherein N-acetyl cysteine, and optionally        curcumin, are incorporated into the cross-linked glucomannan        matrix.    -   3) partially drying the hydrogel obtained in step 2).

A dry xerogel or film matrix can be obtained from a hydrogel by afreeze-drying or convective-drying method according to processes knownto a person skilled in the art. In a preferred embodiment, the dryxerogel is formed from the hydrogel by evaporative-drying process,preferably air-drying, vacuum-drying or convective-drying.

Subsequently, the xerogel is rehydrated to form a hydrogel whichachieves an appropriate release kinetic and, at the same time, a highconcentration of active ingredient(s) are incorporated at the releaseside of the galactomannan matrix.

Finally, the hydrogel is partially drying for its subsequent applicationto the site of interest.

In a particular embodiment of the invention, the hydrogel furthercomprises cells incorporated in the matrix of galactomannan or in thesurface thereof. The incorporation of cells enhances the regenerativeactivity of the hydrogel and the tissue repair process in those tissueshighly damaged or without the possibility of in situ cellularcontribution from the patient, since this biomaterial contains healthycells of the same type as those present in the damaged tissue.

Preferably, the cells incorporated in the hydrogel are selected from thegroup consisting of fibroblasts, keratinocytes, endothelial cells,differentiated or undifferentiated mesenchymal stem cells, cornealcells, epithelial cells, cells from leucocitary system, cells fromhematopoietic system, differentiated or undifferentiated stem cells,chondrogenic cells, osteoblasts, miocytes, adipocytes and neurons orother cells from the peripheric and central nervous system.

In a particular embodiment of the invention, the hydrogel isincorporated in a wound dressing. Therefore, another aspect of thepresent invention refers to a wound dressing which comprises thehydrogel of the invention. The wound dressing is preferably in sheetform and comprises an active layer of the hydrogel according to theinvention. The active layer would normally be the wound contacting layerin use, but in some embodiments it could be separated from the wound bya liquid-permeable top sheet.

The wound dressing may include other ingredients. For example, in orderto decrease the permeability of the wound dressing material, water losscontrol agents may be added. A decrease in the permeability of the wounddressing material controls the loss of fluids form the wound. Preferredwater loss control agents are glycolipids, ceramides, free fatty acids,cholesterol, triglycerides, stearylesters and silicone oil.

If desired, a plasticizer may also be added to the wound dressing. Thepresently preferred plasticizers are glycerol and water, however,propylene glycol and butanol may also be used.

If desired, a hydration control agent may also be incorporated into thewound dressing material. The preferred hydration control agent isisopropyl alcohol, however, ethanol, glycerol, butanol and propyleneglycol may also be used.

Preferably, the wound dressing further comprises a backing sheetextending over the active layer opposite to the wound facing side of theactive layer. Preferably, the backing sheet is larger than the activelayer such that a marginal region extends around the active layer toform a so-called island dressing. In such cases, the backing sheet ispreferably coated with a pressure sensitive medical grade adhesive in atleast its marginal region.

Preferably, the backing sheet is permeable to water vapor, but notpermeable to liquid water or wound exudates. Preferably, the backingsheet is also microorganism-impermeable. This allows the wound under thedressing material to heal under moist conditions without causing theskin surrounding the wound to macerate.

Suitable polymers for forming the backing sheet include polyurethanesand polyalkoxyalkyl acrylates and methacrylates such as those disclosedin GB-A-1280631.

The adhesive (where present) layer should be moisture vapor transmittingand/or patterned to allow passage of water vapor therethrough. Theadhesive layer is preferably a continuous moisture vapor transmitting,pressure-sensitive adhesive layer of the type conventionally used forisland-type wound dressings, for example, a pressure sensitive adhesivebased on acrylate ester copolymers, polyvinyl ethyl ether andpolyurethane as described for example in GB-A-1280631.

The wound facing surface of the dressing is preferably protected by aremovable cover sheet. The cover sheet is normally formed from flexiblethermoplastic material. Suitable materials include polyesters andpolyolefins. Preferably, the adhesive-facing surface of the covers sheetis a release surface. That is to say, a surface that is only weaklyadherent to the active layer and the adhesive on the backing sheet, toassist peeling of the adhesive layer from the cover sheet. For example,the cover sheet may be formed from a non-adherent plastic such as afluoropolymer, or it may be provided with a release coating such as asilicone or fluoropolymer release coating.

Typically, the wound dressing according to the invention is sterile andpackaged in a microorganism-impermeable container.

The hydrogel of the present invention may therefore be used on injuredtissue and for bodily fluid drainages where control and management offluid and secretions is desired. The term “bodily fluid” includes, butit is not limited to, saliva, gingival secretions, cerebrospinal fluid,gastrointestinal fluid, mucous, urogenital secretions, synovial fluid,blood, serum, plasma, urine, cystic fluid, lymph fluid, ascites, pleuraleffusion, interstitial fluid, intracellular fluid, ocular fluids,seminal fluid, mammary secretions, vitreal fluid and nasal secretions.

In particular, the hydrogel is preferably applicable for usage onexudating acute and chronic wounds for controlling accumulating exudatemoisture, support of the wound bed and surrounding tissues.

Accordingly, in an additional aspect, the present invention provides thehydrogel according to the present invention for its use in the treatmentand/or healing of acute surgical and traumatic wounds, burns, scalds,fistulas, venous ulcers, arterial ulcers, pressure sores (otherwiseknown as decubitus ulcers), diabetic ulcers, ulcers of mixed aetiology,and other chronic or necrotic wounds and inflammatory lesions anddisorders.

The hydrogel of the present invention is intended for the treatment ofboth infected and non-infected wounds (that is to say wounds showing noclinical signs of infection). Preferably, the wound is a chronic ornecrotic wound. More preferably, the chronic wound is selected from thegroup consisting of ulcers of venous, arterial or mixed aetiology,decubitus ulcers or diabetic ulcers. Preferably, the hydrogel is used asan antioxidant to reduce oxidative stress in the wound environment andthereby to promote wound healing.

In use, the hydrogel, or the wound dressing containing it, is placed indirect contact with the wound bed. If required, it may be secured intothe position with the wound dressing such as that described above. Ifnecessary, the wound dressing and the hydrogel are removed, whereby anyaccumulated necrotic tissue and exudates is lifted away. The hydrogelmay be replaced by a fresh hydrogel and other suitable wound dressing.

The hydrogel may undergo a swelling action as it absorbs exudatesmoisture, however, they will not dissolve. The swelling action displacesnecrotic material from the wound surface and forces the material intothe matrix of the hydrogel. The laden moisture content and the retentionmoisture near the wound bed by the hydrogel contribute to stimulation ofthe autolytic debridement process whereby the body's own enzymesbreak-up necrotic tissue and cellular debris.

Another aspect of the present invention refers to a cosmetic compositionwhich comprises galactomannan and N-acetyl cysteine.

The cosmetic composition includes any liquid composition or anycomposition which comprises the combination of galactomannan andN-acetyl cysteine and which is in the form of gel, cream, ointment orbalm for its topical administration. Said compositions are characterizedin that they have emollient, protective and healing properties even whenthey do not have any cosmetically active molecule associated.

In a variant of the invention, the cosmetic composition may alsoincorporate active molecules, although they do not have any therapeuticeffect, they have properties as a cosmetic agent. Among the activemolecules which may be incorporated in the antioxidant compositionemollient agents, preservatives, fragrance substances, antiacne agents,antifungal agents, antioxidants, deodorants, antiperspirants,antidandruff agents, depigmenters, antiseborrheic agents, dyes, suntanlotions, UV light absorbers, enzymes, fragrance substances, amongothers, can be cited.

The cosmetic composition may further comprise pH controlling agents,such as, for example, buffer agents, which avoid the pH of thecomposition reducing to values below 5, as well as preservatives whichavoid important structural changes in the composition. A person skilledin the art can determine which additional components can be used and ifthey are necessary, many of them being in common use in cosmeticcompositions.

The cosmetic composition of the invention can be used in the treatmentof an age-related skin damage.

The age-related skin damage refers to any skin condition or disorderassociated with, caused by, or affected by, intrinsic aging and/orextrinsic aging which are often attributed to damage caused by oxygenfree radicals. Oxygen free radicals can damage cells and are believed toaccelerate age-related diseases. Age related skin damage can also becaused by years of sun damage, poor nutrition, high stress levels,exposure to environmental pollution, and certain lifestyles choices,such as cigarette smoking, alcohol or drug abuse.

The aging-related skin condition may, for example, involve wrinkles, agespots, sun damage (particularly UV radiation-induced oxidative stress),blemishes, hyperpigmented skin, increased skin thickness, loss of skinelasticity and collagen content and/or dry skin.

In another aspect, the present invention refers to the use the cosmeticcomposition as described above as an UV-radiation protector.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof.

EXAMPLES Example 1 Estimation of Limit Concentrations of NAC andTurmeric

N-acetyl cysteine (NAC), locust bean gum (LBG) and curcumin (Turmeric orTur) were supplied by Sigma.

The limit concentrations in the use of NAC and Turmeric were establishedafter in vitro cytotoxicity and proliferation assays in humanfibroblasts within a range from 0.5 mM to 20 mM for NAC and from 0.5 μMto 50 μM for turmeric.

The proliferation assay was carried out using the MTT colorimetric assay(Roche 11465007001). MTT is a yellow tetrazolium salt which formsformazan crystals in active cells. The formazan crystals are solubilizedand the resulting color is quantified by means of spectrophotometry at550 nm.

Fibroblasts were seeded in a 96-well plate at a density of 4000 cellsper well. The cells were maintained at 37° C. in an incubation stove.The following day, the treatments of NAC and Turmeric were added to thecell culture using a volume of 200 μL per well. The cell culture wasleft to incubate for 24, 48 and 72 hours.

After each incubation time, 20 μL of MTT (a final concentration of 0.5mg/mL) were added to each well. The plate was maintained for 4 hours inthe incubation stove in order to allow the formation of formazancrystals. Subsequently, 100 μL of solubilizer was added to each well andthe plate was left in the incubation stove until the following day.Then, the data of absorbance at 550 nm were measured.

FIGS. 1a and 2a show the results from cytotoxicity and proliferationassays by means of the MTT colorimetric assay, using differentconcentrations of NAC and turmeric.

The IC₅₀ of each component, i.e. the concentration causing a 50% celldecrease with respect to the control, was established as the toxicitylimit (FIGS. 1b and 2b ).

For the case of NAC, none of the concentrations studied reached IC₅₀ at72 hours, although concentrations of 10 and 20 mM progressively reducedcell proliferation with respect to the control. The maximum limit ofconcentration of NAC could be established at 10 mM. Based on the resultsobtained in the experiments, concentrations of 1 and 5 mM of NAC wereselected since they resulted in an improvement of the proliferative ratein the fibroblasts.

In the case of the study of the maximum limit of concentration ofturmeric, it was observed how the concentrations of 20 and 50 μM havetoxicity from the first 24 hours, exceeding the IC₅₀ limit. Theconcentration of 10 μM exceeds the limit at 72 hours and theconcentration of 7.5 μM reaches the IC₅₀ at 72 hours. Therefore, theconcentration of 7.5 μM can be established as the maximum limit of useof turmeric in the mixture.

Concentrations of 1 and 5 μM of Turmeric were selected for theexperiments since no toxic effects were observed.

Example 2 Effect of the Components of the Composition of the Inventionand the Combination Thereof on the Viability of Human Fibroblasts

The objective of the assay is to determine the effect caused by LBG,NAC, turmeric and combinations thereof on the survival capacity of thecells in an adverse environment, such as the one in the bed of a wound.

To that end, fibroblasts were subjected to an oxidative environmentusing hydrogen peroxide for 1 hour and were put in contact with LBG,NAC, turmeric and combinations thereof. The cell viability offibroblasts in culture was analyzed by means of the MTT colorimetricassay as defined above.

Cell Seeding for the Assay

The day before the assay, the fibroblasts were seeded in a 96-well plateat a density of 11500 cells per well. All the assays were performed intriplicate.

Assay

The treatments were prepared on the day of the experiment and thehydrogen peroxide was added just before the assay.

Preparation of the 1% Locust Bean Gum in Normal Cell Growth Medium

A solution of locust bean gum of 1% in distilled water was prepared andheated above 100° C. until completing dissolution of the gum. Thesolution was then centrifuged for 20 minutes at 4000 rpm to remove theimpurities from the mixture. The solution of the locust bean gum waslyophilized. The liophilisate was dissolved in cell growth medium(DMEM+10% FBS) at a concentration of 1%.

Preparation of the NAC and Curcumin Treatments and of Hydrogen Peroxide

The NAC and curcumin treatments and the hydrogen peroxide were preparedjust before starting the assay. To prepare the curcumin stock, it isnecessary to know the purity of the Turmeric batch available andreadjust the calculation to add the necessary concentration.

The treatments and the hydrogen peroxide were added at the same time andleft to incubate for 1 hour.

After the incubation, the treatments were eliminated from the cells andnormal growth medium and 10% MTT added. The solubilizer was added at 4hours.

Then, the data of absorbance at 550 nm were measured.

To study the synergistic or additive effect of the combination of thecomponents, the results were analyzed by means of applying the formulasspecifically designed to study these parameters:

A. Adapted Formula of the Dose-Modifying Factor (DMF), Referred to asCombination Factor (CF).

The original formula analyzes the dose-modifying factor taking as datathe percentage of cell inhibition caused by two drugs administered aloneand in combination (Thrall B D et al. Differential sensitivities ofmurine melanocytes and melanoma cells to buthionine sulfoximine andanticancer drugs. Cell. Res. 1991; 4: 237-9). Said formula has been usedand published in subsequent international articles of our researchgroup.

The formula presented herein is adapted from the one indicated above,taking as a reference the increase in the percentage of surviving cellswith respect to the oxidized control, and is as follows:

${CF} = \frac{{\% \mspace{14mu} {protection}\mspace{14mu} {LBG}} + {NAC}}{\left( {\% \mspace{14mu} {protection}\mspace{14mu} {LBG}} \right) + \left( {\% \mspace{14mu} {protection}\mspace{14mu} {NAC}} \right)}$${\% \mspace{14mu} {protection}} = {\left\{ {\frac{{Treated}\mspace{14mu} {value}}{{Oxidized}\mspace{14mu} {control}\mspace{14mu} {value}} \times 100} \right\} - 100}$

B. Original Formula Referred to as Combined Index (CI)

The formula presented herein is an original formula of one of theauthors of the patent (T. Palomares) which analyzes the percentage ofsurviving cells in the presence of an agent, alone or in combinationwith others, over the number of original cells by subtracting the numberof surviving cells from the oxidized control. Thus, the increase in thenumber of surviving cells with respect to the cells that are not treatedand exposed to the oxidant is analyzed. The formula is as follows:

${CI} = \frac{\left( {{\% \mspace{14mu} {Cs}\mspace{14mu} {LBG}} + {NAC}} \right) - \left( {\% \mspace{14mu} {CS}\mspace{14mu} {Cox}} \right)}{\left( {{\% \mspace{14mu} {Cs}\mspace{14mu} {LBG}} - {\% \mspace{14mu} {CS}\mspace{14mu} {Cox}}} \right) + \left( {{\% \mspace{14mu} {Cs}\mspace{14mu} {NAC}} - {\% \mspace{14mu} {CS}\mspace{14mu} {Cox}}} \right)}$

Cox: oxidized controlCs: surviving cells with respect to the initial control withoutoxidation

In both formulas, a value>1 indicates a synergistic effect (with highersignificance the greater said value is) and <1 indicates an additiveeffect with has a higher value the closer it is to 1.

Once the appropriate numerical verifications have been made, identicalresults are obtained with both formulas.

The results corresponding to the analysis of the experiments in whichthe cells were subjected to an oxidative environment (1 mM H₂O₂) andtreated with 1% LB G, 1 mM NAC, 1 mM Turmeric and combinations thereofare shown in FIG. 3. The experimental data were obtained 1 hourpost-oxidation. These data indicate that the triple combinationLBG+NAC+Tur produces the best protection effect, reaching controllevels. The combination LBG+NAC increase also the cell viability withrespect to oxidized control and with respect to LBG, NAC, Tur, LBG+Turand NAC+Tur treatments.

Table I shows the percentage of viability of the cells subjected tooxidative stress with respect to the non-oxidized control group.

Oxidation 1 mM H₂O₂ TREATMENTS Absorb % Viability Control 0.18 100Oxidation control 0.09 48.8 1% LBG 0.1 56.59 1 mM NAC 0.1 57.14 1% LBG +1 mM NAC 0.14 77.77 1 μm Turmeric 0.12 63.18 1 mM NAC + 1 μm Turmeric0.12 64.28 1% LBG + 1 mM NAC + 1 μm Turmeric 0.2 100

Table II shows the indices obtained by means of applying formulas A andB in which it is concluded that there is a synergistic effect in thecombinations of LBG+NAC and the triple combination of LBG+NAC+Turmeric.

Treatment Value Effect 1% LBG + 1 mM NAC 1.69 Synergistic 1% LBG + 1 μMTur 0.75 Additive 1 mM NAC + 1 μMTur 0.67 Additive 1% LBG + 1 mM NAC + 1μm Tur 1.85 Synergistic

The results have pointed out that the combinations of LBG, either withNAC or with NAC+Tur, cause a synergistic effect in the increase of cellviability in a oxidative stress situation. However, the combination ofLBG+Tur and NAC+Tur produces the expected additive effect.

The analysis of the most pronounced protection effects show that thecombination of the three agents results in the greatest protectiveeffect (100% of surviving cells). The combination of the three agentsshows an effect that is 1.3 times greater than the treatment withLBG+NAC (77.77%).

Example 3 Effect of the Components of the Composition of the Invention,Alone or in Combination, on the Decrease of the Oxygen ReactiveMetabolites Generated in the Human Fibroblasts Subjected to an OxidativeEnvironment

The increase of reactive oxygen metabolites (ROMs) is one of the maincauses hindering the healing of a wound. This effect contributes to theloss of proliferative capacity of the cells and to the increase in theexpression of metalloproteases, which degrade the new dermal matrixformed and prevent healing.

To quantify the ROM-decreasing or antioxidant capacity of LBG, NAC andturmeric, the production of the ROMs generated upon oxidizing a cultureof fibroblasts with a high concentration of hydrogen peroxide wasmeasured.

The intracellular ROMs were quantified by means of the labeling thereofwith the fluorescent probe 2′,7′-dichlorofluorescein diacetate(Molecular Probes D399). This probe is capable of emitting fluorescenceat 538 nm when it is oxidized with reactive oxygen metabolites. The celloxidation was carried out with 1 mM hydrogen peroxide.

Cell Seeding for the Assay

The day before the assay, the fibroblasts were seeded in a 96-well plateat a density of 11500 cells per well. All the assays were performed intriplicate.

Assay

The treatments were prepared on the day of the experiment and thehydrogen peroxide was added just before the assay.

Preparation of the 1% Locust Bean Gum in Normal Cell Growth Medium

A solution of locust bean gum of 1% in distilled water was prepared andheated above 100° C. until completing dissolution of the gum.

The solution was then centrifuged for 20 minutes at 4000 rpm to removethe impurities from the mixture. The solution of the locust bean gum waslyophilized. The lyophilisate was dissolved in cell growth medium(DMEM+10% FBS) at a concentration of 1%.

Labeling the Cells with the Fluorescent Probe

Before adding the treatments and the hydrogen peroxide, the cells werelabeled with the fluorescent probe at a concentration of 50 μM for 30minutes in darkness.

Preparation of the NAC and Turmeric Treatments and of Hydrogen Peroxide

The NAC and Turmeric treatments and the hydrogen peroxide were preparedjust before starting the assay. To prepare the turmeric stock, it isnecessary to know the purity of the Turmeric batch available andreadjust the calculation to add the necessary concentration.

After labeling the cells, the antioxidant treatments and the hydrogenperoxide were added.

The fluorescence emitted at 538 nm by the probe was collected 20 minutesafter the start of the oxidation.

FIG. 4 shows the intracellular ROM levels of the fibroblasts subjectedto an oxidative environment using 1 mM of H₂O₂, by means of thefluorescence units obtained in the labeling with the probe2′,7′-dichlorofluorescein diacetate and also when fibroblasts are put incontact with 1% LBG, 5 mM NAC, 5 μM Turmeric and combinations thereof.

Table III shows the data of the percentage of decrease of ROMs withrespect to the oxidized control, when the cells were subjected to 1 mMof hydrogen peroxide and in contact with the components of thecomposition of the invention.

1 mM H₂O₂ F.U. % decrease of ROS CONTROL 0.157 OXIDIZED CONTROL 12.47LBG 1.74 86 NAC 1.82 85 Tur 2.31 81 NAC + Tur 0.97 92 LBG + Tur 1 91LBG + NAC 0.307 97 LBG + NAC + Tur 0.157 99

As can be observed, there is a significant decrease of the intracellularROM levels in the cells which are in contact with LBG, NAC, Turmeric andcombinations thereof.

The addition of NAC to the solution of LBG causes a significant decreaseof the intracellular ROM levels with respect to the LBG alone.

However, the triple combination LBG+NAC+Tur produces the highest benefitin terms of the decrease of the intracellular ROM levels, which aresimilar to those of the control group (without oxidation).

In order to verify the synergistic or additive effect of the combinationof the different components, the results were analyzed by applyingformulas A and B mentioned in example 2. However, in this case thedecrease in intracellular ROM levels was taken as a reference withrespect to the oxidized control.

The results are shown in Table IV:

Treatment Value Effect LBG + NAC 3.29 Synergistic LBG + Tur 0.65Additive NAC + Tur 0.68 Additive 1% LBG + 1 mM NAC + 1 μM Tur 3.30Synergistic

The application of formulas A and B show clearly a synergistic effectproduced by the LBG+NAC and LBG+NAC+Tur combinations, whereas theLBG+NAC and NAC+Tur combinations produce and additive effect, withrespect to intracellular ROS reduction.

Example 4 Preparation of a Hydrogel of Locust Bean Gum with N-AcetylCysteine Incorporated Therein

A weighed amount of locust bean gum was dispersed in distilled water toform a solution containing 1-5 wt % of said gum. In order to favor thesynthesis of the hydrogel, sulfuric acid was added to the solution untilobtaining a pH of 2, with the aim of protoning hydroxyl groups of thelocust bean gum. The solution was stirred at room temperature for 2-3hours and, subsequently, the temperature was raised until 100-120° C. Atthis temperature, the solution was stirred for at least 30 minutes.

The solution was centrifuged at 4000 rpm for 20 minutes in order toremove impurities in the mixture, thus the pure locust bean gum solutionis in the supernadant and the impurities are deposited in the pellet.

The locust bean gum solution was subjected to a chemical cross-linkingstep using glutaraldehyde as cross-linking agent. For this purpose,glutaraldehyde was added to the solution of locust bean gum whilestirring for at least 30 minutes. The amount of glutaraldehyde dependson the desired final characteristics of the hydrogel. If a quickdelivery of the N-acetyl cysteine is required, lower quantities ofcross-linking agent are added to the solution of locust bean gum inorder to obtain a low cross-linking degree. On the contrary, if anincreased delivery time of N-acetyl cysteine is required, highquantities of cross-linking agent are added to the solution of locustbean gum in order to obtain a high cross-linking degree. FIGS. 5a-5dcorrespond to photographs taken from scanning electron microscope (SEM)which show the increase in the porosity degree of a hydrogel of locustbean gum at 3% by weight when increasing the concentration of thecross-linking agent from 0 to 2.5% by weight.

The mixture of locust bean gum and glutaraldehyde was placed on petridishes. The cross-linking reaction was carried out at 37° C.

Once the hydrogel has been formed, it was washed with sodium bisulfate(Sigma 13438) at 5% and then with distilled water, in order to removethe non-reacted glutaraldehyde. Subsequently, the hydrogel was dried inan oven at 65° C. to form a xerogel.

In order to incorporate the N-acetyl cysteine into the structure of thelocust bean gum, the xerogel was rehydrated by introducing it into asaturated solution of N-acetyl cysteine and PBS. Finally, the obtainedhydrogel was partially dried for its subsequent use.

Example 5 Assessment of the Effect of a Hydrogel Containing LBG,LBG+NAC, and LBG+NAC+Tur, on the Wound Healing Process in the Pig Skin

Four male pigs of 25-35 kg body weight were selected. Before startingthe procedure, the animals were submitted to 1 week acclimatizationperiod.

Preoperatively, the animals were sedated with intramuscularly azaperone(4 mg/kg)+ketamine (10 mg/kg) and tracheally intubated and analgesia wasinduced with intravenous buprenorfine (0.01 mg/kg). Anaesthesia wasinduced and maintained with propofol (4 mg/kg), isoflurane (1.5-2%,oxygen). Presurgical antibiotherapy was performed with intravenouscephalotin (22 mg/kg).

Four skin lesions were surgically generated in the dorsal area of eachpig. Three different biomaterial matrix combinations (LBG, LBG+NAC andLBG+NAC+Tur) were applied in three of the four lesions and saline wasapplied in the control lesion. The dressings were replaced every 3 dayswhen wounds were cleaned and dressing changed.

In the postoperative period, a macroscopic evaluation of tissue healingwas performed throughout the experiment. Biopsies for histologicalevaluation were obtained 5, 10 and 15 days after lesions generation. Allskin biopsy samples were fixed in 10% neutral buffered formalin,routinely processed and stained with hematoxylin and eosin (H&E) forhistopathological study. Histopathological evaluation in treated andcontrol areas was performed. Parameters such as epidermisreepitelization, presence of dermal inflammation and fase of granulationtissue formation and maduration were assessed.

The results show the faster evolution from the initial days, mainly inLBG+NAC and LBG+NAC+Tur treated lesions. Furthermore, the microscopicanalysis showed an improvement in the granulation tissue formation andin the maduration of this tissue with respect to control lesion.

FIG. 6 shows a macroscopic view of a 10 days of evolution biopsy,wherein it can be observed an increase in the new tissue formation inthe treated groups, but particularly in the group treated with aLBG+NAC+Tur.

When calculations of wound surface were performed in the three groups,an improvement in the wound closure capacity was shown in the threegroups. However, this effect was increased in LBG+NAC group and thehigher effect was observed in the LBG+NAC+Tur group.

Table V shows the estimated lesion area in the different treated groupsand the index that indicates the capacity of wound reduction presentedby these groups. The index also indicates that the higher effect wasachieved by LBG+NAC+Tur treatment.

Lesion area reduction index Lesion area (relative to (cm²) controlgroup) Control 7 1 LBG 5.88 1.2 LBG + NAC 4.62 1.5 LBG + NAC + Tur 3.781.85

FIG. 7 shows a three days evolution photograph where treatments with LBG+NAC and LBG+NAC+Tur were compared with an established collagentreatment. As can be observed, there is a reduction in lesion area inboth LBG+NAC and LBG+NAC+Tur groups with respect to control and collagentreated group, and again the LBG+NAC+Tur presented the higher areareduction and the best quality healing process.

1-22. (canceled)
 23. A method for therapeutic or prophylactic treatmentof a skin disease or condition resulting from reactive oxygen speciesproduction in the skin of a subject or of a skin disease or conditionwhich involves reactive oxygen species production in the skin of asubject, the method comprising topical administration of atherapeutically effective amount of a composition comprisinggalactomannan and N-acetyl cysteine.
 24. The method according to claim23, wherein the galactomannan comprises locust bean gum.
 25. The methodaccording to claim 23, wherein the reactive oxygen species productionresults from exposure of the subject to sunlight radiation, chemicalagents, radiotherapy or chemotherapy.
 26. The method according to claim23, wherein the skin disease or condition resulting from reactive oxygenspecies production in the skin of a subject is selected from the groupconsisting of sunburn, photosensitivity, immunosuppression, prematureaging, psoriasis, an immunological disease, a localized or widespreadinflammation, a bacterial or fungal infection, skin rashes, systemicoxidative stresses, actinic keratosis, and skin cancer selected from thegroup consisting of basal cell cancer, squamous cell cancer, andmalignant melanoma.
 27. The method according to claim 23, wherein skindisease or condition which involves the reactive oxygen speciesproduction in the skin of a subject is selected from the groupconsisting of acute surgical and traumatic wounds, burns, scalds,fistulas, venous ulcers, arterial ulcers, pressure sores, diabeticulcers, ulcers of mixed aetiology, chronic or necrotic wounds,inflammatory lesions, and inflammatory disorders.
 28. The methodaccording to claim 23, wherein the composition further comprisescurcumin as an additional antioxidant ingredient.
 29. A hydrogelcomprising galactomannan and N-acetyl cysteine, wherein thegalactomannan is in the form of a cross-linked matrix, and the N-acetylcysteine is incorporated in said cross-linked matrix of galactomannan.30. The hydrogel according to claim 29, wherein the galactomannancomprises locust bean gum.
 31. The hydrogel according to claim 29,wherein the cross-linked matrix of galactomannan further comprisescurcumin incorporated therein.
 32. The hydrogel according to claim 29,further comprising cells.
 33. The hydrogel according to claim 32,wherein the cells are selected from the group consisting of fibroblasts,keratinocytes, endothelial cells, differentiated or undifferentiatedmesenchymal stem cells, corneal cells, epithelial cells, cells fromleucocitary system, cells from hematopoietic system, differentiated orundifferentiated stem cells, chondrogenic cells, osteoblasts, miocytes,adipocytes, and neurons or other cells from the peripheric and centralnervous system.
 34. A process for preparing a hydrogel as defined inclaim 29, the process comprising: a) dissolving the galactomannan in anaqueous solution; b) subjecting the galactomannan to a chemicalcross-linking by adding a cross-linking agent to the aqueous solution ofgalactomannan to obtain a hydrogel comprising a cross-linked glucomannanmatrix; and c) incorporating N-acetyl cysteine into the cross-linkedglucomannan matrix.
 35. The process according to claim 34, wherein theincorporation of N-acetyl cysteine into the cross-linked glucomannanmatrix comprises the following steps: 1) drying the hydrogel obtained instep b) to form a xerogel; 2) rehydrating the xerogel by introducing thexerogel in an aqueous solution comprising N-acetyl cysteine to form ahydrogel wherein N-acetyl cysteine is incorporated into the cross-linkedglucomannan matrix; and 3) partially drying the hydrogel obtained instep 2).
 36. The process according to claim 34, further comprisingincorporating curcumin into the cross-linked glucomannan matrix.
 37. Theprocess according to claim 36, wherein the incorporation of curcumininto the cross-linked glucomannan matrix comprises the followingsteps: 1) drying the hydrogel obtained in step b) to form a xerogel; 2)rehydrating the xerogel by introducing the xerogel in an aqueoussolution comprising curcumin to form a hydrogel wherein curcumin isincorporated into the cross-linked glucomannan matrix; and 3) partiallydrying the hydrogel obtained in step 2).
 38. A method for treatmentand/or healing of acute surgical and traumatic wounds, burns, scalds,fistulas, venous ulcers, arterial ulcers, pressure sores, diabeticulcers, ulcers of mixed aetiology, or other chronic or necrotic woundsand inflammatory lesions and disorders, the method comprising topicaladministration of a therapeutically effective amount of a hydrogel asdefined in claim
 29. 39. A wound dressing comprising a hydrogel asdefined in claim
 29. 40. A method for treatment of age-related skindamage, the method comprising topical administration of a cosmeticcomposition comprising galactomannan and N-acetyl cysteine.
 41. A methodfor protecting skin from damage inducible by UV-radiation, the methodcomprising topical administration of a cosmetic composition comprisinggalactomannan and N-acetyl cysteine.